Organisms encode multiple Hsp70s to increase capacity to regulate abundance of Hsp70 in accordance with need and to provide a range of distinct Hsp70 functions for carrying out specific tasks within all cells or in distinct cell types. We constructed a yeast system to evaluate Hsp70s from any source and are using it to investigate how Hsp70s within and across species influence propagation of amyloid in vivo and act in cellular protein quality control (PQC). The wide range of responses that prions have to alterations of Hsp70s and their co-chaperones provides a sensitive way to investigate even subtle functional distinctions among highly redundant Hsp70s and an approach to uncover the underlying mechanisms. Hsp90 is another major PQC chaperone that has essential roles in many fundamental cellular processes. Its functions rely on interaction and cooperation with the Hsp70 system and its own co-chaperones. We earlier identified a single Ala/Gly difference in the nearly identical Hsp70s Ssa1 and Ssa2 as solely responsible for differential ability of Ssa1 and Ssa2 to support propagation of URE3 prions. We further showed this tiny difference also determined functional specificity of Ssa1 and Ssa2 in a cellular protein degradation pathway important for regulating abundance of gluconeogenic enzymes. Faulty regulation of these enzymes can cause diabetes. Our findings imply that regulation of Hsp70 substrate binding, not substrate binding per se, can underlie functional specificity. A large cohort of co-chaperones regulate and fine-tune Hsp70 substrate binding activity. Because intrinsic enzymatic activities of Ssa1 and Ssa2 are similar, we suspect that Hsp70 co-factors are responsible for the differences in how they function in protein degradation and we are working to identify them. J-proteins are obligate and highly abundant Hsp70 co-chaperones. Humans have a similar number of cytosolic Hsp70s as yeast, but over twice as many J-proteins. Thus, while intrinsic Hsp70 activities have diversified little, the ability of Hsp70 to perform additional or more complex tasks in humans has expanded, in part, by amplification of J-proteins. The extent to which J-proteins specify Hsp70 functions by providing their own unique substrate interactions or by their ability to recruit and regulate basic Hsp70 activities is far from understood and we are working to gain more insight into these processes. We also are working to determine if differences in functions of Hsp70s are mediated by ways they cooperate with J-proteins, other co-chaperones or other major chaperones, such as Hsp90. We are working to learn whether such differences in Hsp70 function contribute to protection from amyloid toxicity that we see in some of our strains and if human chaperones possess such protective functions. Another difference between Ssa1 and Ssa2 is that increasing expression of Ssa1, but not Ssa2, cures wild type cells of URE3. This difference is not determined by the Ala/Gly variation at position 83 that is responsible for the difference in the ways these Hsp70s support URE3 propagation. We are working to identify the structural and functional differences between Ssa1 and Ssa2 to determine how these Hsp70s differ in their anti-prion effects. Altering abundance or function of Hsp70 or Hsp90 can moderate pathology in models of protein folding disorders, while in the same models reducing chaperone activity can exacerbate, or alone even cause pathology. Hsp70 and Hsp90 therefore are promising therapeutic candidates for amyloid and other protein folding disorders and they are being evaluated extensively as drug targets. Altering Hsp70 and Hsp90 co-chaperones also moderates pathology in several models of amyloid and other protein folding disorders. Our findings can help guide decisions about which Hsp70/90-family members would be most useful for such applications, or identify potential problems that could arise due to distinct sensitivities of different Hsp70s or Hsp90s to specific compounds. Overall our work provides insight into functions of these chaperones that can help guide strategies for using chaperones as targets for therapy in such diseases.